Methods of treating nerve-related vision disorders by an insulinomimetic agent

ABSTRACT

This invention provides reagents and methods for delivering insulin, insulinomimetic agents, and the like to a vertebrate eye via subconjunctival routes, sub-Tenon&#39;s routes, or intravitreal routes for treatment of nerve-related vision disorders such as diabetic retinopathy, and formulations useful in the practice of the disclosed methods.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/755,232, filed May 30, 2007, now U.S. Pat. No. 7,829,532, which is acontinuation of U.S. patent application Ser. No. 10/375,973, filed Feb.28, 2003, now U.S. Pat. No. 7,247,702, which claims the benefit of U.S.Provisional Application Ser. No. 60/361,559, filed Feb. 28, 2002, theentire content of all of which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to a method for delivering insulin,insulinomimetic agents, and the like to the eye for treatment ofdiabetic retinopathy. More specifically, the method involves theperiocular administration of these drugs via subconjunctival routes,sub-Tenon's routes, or intravitreal routes.

BACKGROUND OF THE RELATED ART

Diabetes has reached epidemic proportions. Approximately 15 millionpeople in the United States are currently afflicted with the disease,and that number is expected to rise to at least 21 million over the next30 years.

In addition to (and as a consequence of) the metabolic disarray causedby the disease, diabetes causes a variety of other, organ-specificdysfunctions, including in particular diabetic retinopathy. Diabeticretinopathy affects half of all Americans diagnosed with diabetes.Diabetic retinopathy is an illness that occurs when diabetes damagestiny blood vessels in the retina, affecting vision, and is a leadingcause of blindness. There are two clinical stages of retinopathy. Thefirst stage is known as nonproliferative retinopathy, in which the bloodvessels damaged by diabetes leak fluid and lipids onto the retina. Whenthe fluid accumulates in the center of the retina (i.e., the macula) itleads to macular edema. The fluid makes the macula swell, which blursvision. The second stage is the proliferative stage, where new bloodvessels grow along the retina and in the clear, gel-like vitreous thatfills the inside of the eye. These new blood vessels can bleed, cloudvision, and destroy the retina unless treated. There is also apreclinical phase in which patients will generally have no symptoms, norwill there be any findings on routine clinical examination. However, inthe preclinical phase sensitive tests reveal reduced contrastsensitivity, electrical responses with an electroretinogram, or colorvision.

There are several methods of treatment for diabetic retinopathydisclosed in the art. However, none of these treatment approaches haveproven successful in addressing the primary metabolic disorder or inpreventing retinopathy. Conventional diabetic retinopathy treatments arelimited to controlling the diabetic state with systemic insulinadministration or oral hypoglycemic agents. The problem with thesesystemic approaches is that they do not restore normal physiologicmetabolic control or provide overall effective levels of the drug to theeye. Secondary treatment approaches include using diuretics to controlblood pressure or intravascular fluid overload. Attempts have also beenrecognized in the arts for treating retinopathy with aldose reductaseinhibitors, inhibitors of nonenzymatic glycation (aminoguanidine),corticosteroids or antihistamines. Methods of treatment for advancedretinopathy complications include vitrectomy surgery and lasertreatment, exposing an intense beam of light to the small diseased areasof the retina. These methods are palliative in nature, and none of thesemethods is sufficiently effective to prevent or cure the disease.

Although diabetic retinopathy is extensively studied in the art, thedirect effects of insulin or insulinomimetics on diabetic retinopathyare limited. It has been demonstrated that retinal neurons die inexperimental diabetes in rats and in humans. Moreover, insulin has beenshown to be a survival factor for retinal neurons in culture, and excesshexosamines impair insulin's survival-promoting effects. In vivo,systemically and intraocularly administered insulin activates theinsulin receptor and downstream signaling cascades that are involved incell function and survival. However, the ability to administersystemically sufficient insulin or other insulinomimetic agents to beeffective for prevention of retinopathy is limited by the risk ofhypoglycemia.

Accordingly, there is a great demand for safe and effective methods fordelivering agents effective in treating diabetic retinopathy. Inparticular, there is a need in the art for treatment methods thatmaintain retinal cell function and survival in the face of persistenthyperglycemia.

SUMMARY OF THE INVENTION

The invention describes methods and reagents for treating retinaldisorders, particularly retinal disorders having at least in part ametabolic etiology. As provided herein, the inventive methods andreagents permit compounds for treating ocular disorders, such as retinaldetachment, retinitis pigmentosa, central retinal artery occlusion,central retinal vein occlusion, ischemic optic neuropathy, high tensionglaucoma, low tension glaucoma, and cataract, to be administered locallyin the eye. The invention specifically provides methods for preventingand treating nerve-related vision disorders, including in particulardiabetic retinopathy. The inventive methods comprise periocularadministration of a sufficient amount of a drug by a subconjunctival,sub-Tenon's or intravitreal route to be effective in treating suchretinal disorders. In certain embodiments, the drug is administered toan eye under its mucous membrane or fascia.

Preferred drugs administered using the methods of the invention includeformulations of insulin, insulinomimetic agents, or peptides.Formulations of insulin that may be used in the invention include, forexample, formulations of native insulin, naturally derived insulin,recombinant insulin, any modification thereof containing buffers ormodifying proteins, or any other known formulations of insulin. Theconcentration of the insulin formulation can range from 1 picomolar to100 micromolar. If the insulin formulation is a gel or liquid, thevolume thereof can range from 5 μL to 1 mL. In the practice of theinventive methods, a sufficient amount of any of these drugs isadministered to the eye, wherein a sufficient amount of insulin rangesfrom 5 to 100 or 0.1 to 10 units of insulin. Formulations can alsoinclude augmenting drugs from the thiazolidinediones (TZD) class, suchas rosiglitazone, pioglitazone, and troglitazone, as well as,non-peptide insulinomimetic agents, such as TLK16998 (Telik), KRX-613,and L-783,281 (Merck).

The invention further comprises methods of administrating drugs to theeye, where drugs are administered to more than one eye. These methods ofadministration include via a solution, a polymeric base, or a pump.Additionally, the method of administration may be by implanting adevice, where the device releases a formulation of a drug, preferablyinsulin, an insulinomimetic agent or a peptide at a prescribed rate. Oneor more devices may be administered to one eye.

The invention also provides formulations of insulin, insulinomimeticagents or peptides adapted or prepared for use with the methods of theinvention. Preferably, the formulations of the invention comprisepharmaceutical adjuvants, carriers, buffers or other components.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the invention's method for administration of drugsunder the eye's surface membrane;

FIG. 2A presents a phosphotyrosine (PY) detecting Western blot from bothmuscle and retina in a rat that received either a vehicle (V) andinsulin (I) injection;

FIG. 2B presents a graph quantifying Western blot analysis andexpressing data as a graph for muscle tissue in terms ofphosphotyrosine/insulin receptor beta-subunit (PY/IRβ) ratios;

FIG. 2C presents a graph quantifying the Western blots and expressingdata as a graph for retina tissue in terms of PY/IRβ ratios;

FIG. 2D presents an immunoblot of phospho-Akt^(ser473) (top) and totalAkt (bottom) from vehicle, and insulin injected rats as well asquantification of the blot expressed in terms of phosphorylated to totalAkt ratios;

FIG. 3A presents a Western blot comparing IRβ tyrosine phosphorylationin the retina and in other insulin responsive tissues under fasted andfreely fed conditions;

FIG. 3B presents a graph quantifying Western blot analyses as disclosedabove and expressed as a graph of PY/IRβ ratios for retina tissue;

FIG. 4 presents a PY immunoblot for autophosphorylation with (+) andwithout (−) addition of ATP to the kinase reaction in the retina andliver under freely fed and fasted conditions;

FIG. 5 presents a graph quantifying the Western blots shown in FIG. 4and expressed as a graph of IRβ activity when IRβ immunoprecipitateswere subjected to kinase assays;

FIG. 6 presents a PY immunoblot for retinas treated with IGF-1 orinsulin and subjected to immunoprecipitation of the IGF-1Rβ complex,with the arrowhead indicating the tyrosine phosphorylated band thatcorresponds to the IGF-1Rβ complex;

FIG. 7 illustrates the difference between intraportal insulin injectionand explanted retina tissue; and

FIG. 8 presents a graph quantifying an immunoblot of insulin receptortyrosine phosphorylation from retina of an anesthetized rat thatreceived insulin in one eye and vehicle in the other eye.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides reagents, pharmaceutical compositions and methodsfor delivering drugs for treatment or prevention of nerve-related visiondisorders, particularly diabetic retinopathy. The method specificallycomprises periocular administration of insulin, insulinomimetic agentsor peptides via subconjunctival, sub-Tenon's or intravitreal routes.

More specifically, the method of the invention comprises periocularlyadministering a sufficient amount of a formulation of insulin,insulinomimetic agents or peptides to both eyes. Periocular delivery issafer for the general health of an animal, preferably a human,undergoing treatment for a nerve-related vision disorder since itinvolves less ocular morbidity than laser or vitrectomy surgery.

The ability to systemically administer sufficient amounts orconcentrations of insulin or other insulinomimetic agents to preventretinopathy is largely limited by the risk of hypoglycemia. Theinvention overcomes the consequences of systemic administration byachieving direct local drug delivery of effective amounts of theseagents by direct administration to the subconjunctiva, sub-Tenon'scapsule, or intravitreous. Administering insulin or insulinomimeticagents via these routes also provides effective intraocular drugpenetration to maintain retinal cell function and survival.

One aspect of this invention involves replacing a deficient insulinreceptor ligand and increasing activation of a down-regulated insulinreceptor, or its downstream signaling molecules. Specifically, theinventive methods comprise direct insulin administration to the eye, andspecifically to cells of the retina. Any pharmaceutically acceptableinsulin formulation can be used with the methods of the invention.Examples of useful insulin formulations include native insulin(preferably human insulin, particularly recombinantly-produced humaninsulin such as Humulin®, or insulin isolated from any other mammalianspecies), naturally derived or recombinant, and all of modificationsthereof, such as Regular to NPH, Ultralente (Eli Lilly & Co.), insulinglargin (Lantus®, Aventis), Lispro®, (Eli Lilly & Co.), Novolin®(Novo-Nordisk) and formulations containing any modifying proteins (suchas, for example, protamine) or buffers known or accepted in the art.

Moreover, the invention provides methods comprising administration ofinsulinomimetic agents or nucleotides (aptamers) that mimic some or allof insulin's actions. The invention further encompasses theadministration of drugs that augment insulin along with the insulin.These augmenting drugs can be, inter alia, from the thiazolidinediones(TZD) class. They may also be small non-peptide insulinomimetic agentssuch as TLK16998 (Telik), KRX-613, and L-783,281 (Merck). Such compoundsactivate the proliferator-activator gamma (PPAR-gamma) receptor toprovide necessary actions of insulin in the retina. Thus, their additionenhances the insulin effect on signaling to retina cells. Examples ofaugmenting drugs include rosiglitazone, pioglitazone, and troglitazone.

Concentrations of the drugs used in the invention can range from lowpicomolar to micromolar concentrations. If the drug is a liquid or gelinsulin formulation, volumes can range from about 10 μL to about 1 mL. Asufficient dosage of the insulin will range from a few picomolars tomicromolar.

The inventive treatment provided herein permits a number of differentadministration routes to be used to introduce an effective amount of adrug to the eye. These include administering the drug via a pump, apolymeric base, or a solution. The preferred method of administration isby a polymeric base, including but not limited to polyester (PET),polyethylene (PE), poly(L-lactic acid) (PLA), and polyurethane.Additionally, drugs may be administered by implantation of a formulationof the invention or a device that will release such a formulation at aprescribed rate. The invention advantageously provides methods foradministering said formulation to both eyes simultaneously, althoughembodiments having administration to one eye, as well as embodimentshaving independent or non-contemporaneous administration to both eyes,are also encompassed by the invention.

Drug formulations of the invention advantageously can be administeredunder the mucous membrane of the eye or the Tenon's fascia of the eye.More specifically, the drugs can be delivered to the subconjunctivaland/or sub-Tenon's space. As shown in FIG. 1, drug formulations of theinvention are injected, or otherwise administered, under the eye'ssurface membrane so that the drugs are able to diffuse through thesclera into the retina, vitreous, and the anterior chamber.

The inventive methods for treatment of nerve-related retinal disorders,such as diabetic retinopathy, are suitable for prevention or treatmentat any stage of such retinopathic disorders. Specifically, the inventivemethods are equally effective for the preclinical, nonproliferative,macular edema stages of such retinopathic disorders, as well as for theproliferative stage of retinopathy. Other retinal disordersadvantageously treated using the methods of the invention, includeretinal detachment, glaucoma, retinitis pigmentosa, central retinalartery or central retinal vein occlusion, ischemic optic neuropathy,high tension glaucoma, low tension glaucoma, and cataract.

While the invention has been described with particular reference todiabetic retinopathy treatment and other retinal disorders, it will beunderstood by those skilled in the art that the invention hasapplications in other medical fields, in particular whenever localinsulin or insulinomimetic agents are delivered to tissues at risk forcomplications. For example, deliveries to kidneys and nerves sincepatients with diabetes have impaired kidney function (nephropathy) andnerve function (neuropathy). Thus, local application of insulin adjacentto these organs and tissues may improve their function and preventfuture deterioration.

The following Examples are intended to further illustrate certainaspects of the above-described method and advantageous results. Thefollowing examples are shown by way of illustration and not by way oflimitation.

Example 1

Intraportal insulin injection was performed to determine if a singlebolus insulin injection, and therefore an acute elevation in circulatinginsulin, could activate retinal insulin receptors (IR) in vivo as itdoes in other tissues. Intraportal insulin injection was conducted asfollows. Male Sprague-Dawley rats (Charles River, Mass.) weighing 200350 g were fasted 18 hours prior to being anesthetized with a 10:1ketamine:xylazine cocktail (53.5 mg/kg ketamine and 5.33 mg/kg xylazine)administered by intramuscular injection. The fasted rats wereadministered a 500 μg bolus of insulin, nothing (sham), or vehicle (0.9%saline) via the portal vein. At 15, 30, 45 and 60 minutes post injection(shown in FIG. 2A), hindquarter skeletal muscle and retina weresnap-frozen under liquid nitrogen, and then stored at −80° C. pendinganalysis by immunoprecipitation and immunoblotting. There was nodifference in insulin receptor beta-subunit (IRβ) phosphorylationbetween vehicle (V) and insulin (I) injection.

Tissue lysates were immunoprecipitated and immunoblotted as described byBarber et al. (2001, J. Biol. Chem. 276: 32814 32831). Tissues werehomogenized in an immunoprecipitation (IP) lysis buffer (consisting of50 mM HEPES, pH 7.3, 137 mM NaCl, 1 mM MgCl₂, 2 mM NaVO₄, 10 mMNa₂H₂P₂O₇, 10 mM NaF, 2 mM EDTA, 2 mM PMSF, 10 mM benzamidine, 10%glycerol, 1% NP-40, and 1 protease inhibitor tablet(Boehringer-Mannheim) per 10 mL of buffer. Homogenates were rocked 15minutes at 4° C. and then centrifuged at 14,000 rpm at 4° C. Prior toimmunoprecipitation and immunoblotting, the resulting supernatant wassubjected to protein assay (Bio-Rad) and quantification.

Immunoprecipitations were performed as follows. Protein (250 μg) wasdiluted into 1 mL IP buffer containing one of the following antibodies:5 μL of anti-IRβ or anti-IGF-IRβ (Santa-Cruz, Santa Cruz,), or 4 μL ofanti-IRS-1 or IRS-2 (Upstate Biotechnology, Lake Placid, N.Y.), specificfor these species of insulin responsive substrate, and 30 μL of a 50%protein A/Sepharose bead slurry (Amersham Pharmacia Biotech, Piscataway,N.J.). The Sepharose bead complex was rocked overnight at 4° C., washedtwice with 200 μL of IP buffer, and boiled with 30 μL of 2× samplebuffer (a solution of glycerol, SDS, TRIS buffer, bromophenol blue andbetamercapto-ethanol). Fifty μg of protein per sample were analyzed bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). Thereafter,gel-separated proteins were transferred to nitrocellulose membranes(blocked with a solution of 0.05% Tween-20 and 5% non-fat milk or 3%bovine serum albumin, dissolved in Tris-buffered saline) at roomtemperature for 1 hour. The membranes were probed overnight in blockingsolution at 4° C. at 1:1000 dilutions of the primary antibody. Theprimary antibodies used in these assays included an anti-phosphotyrosineantibody (Upstate Biotechnology), and anti-Akt antibodies (CellSignaling, Beverly, Mass.). Secondary antibodies were diluted 1:4000(for horseradish peroxidase-conjugated anti-rabbit antibodies; obtainedfrom Amersham Pharmacia Biotech, Piscataway, N.J.), or 1:1000 (forbiotin-conjugated anti-mouse antibodies; obtained from AmershamPharmacia Biotech). Tertiary incubations with streptavidin-linkedalkaline phosphatase were diluted 1:4000 (Gibco, Gaithersburg, Md.).Positive signals were detected with electrochemiluminescence (ECL) kits(Cell Signaling) and electrochemifluoresence (ECF) kits (Amersham) eachperformed according to each manufacturer's protocol. Immunoblotquantitation was accomplished using ImageQuant (Molecular Dynamics), NIHImage 1.6 (NIH), or GeneTools (SynGene) software. Blots werere-incubated with different antibodies after being stripped, at 50° C.for 1 hour in a buffer containing 63 mM Tris (pH 6.8), 2% SDS, and 0.07%2-mercapthoethanol.

Western blots interrogated with anti-phosphotyrosine (PY) antibodiesshowed a robust response in muscle, as expected. IRβ phosphorylation wasalso increased in retinal tissue, but only after a 30 minute delay. Inaddition, the response had a smaller magnitude than muscle. Nodifference in IRβ phosphorylation was found in sham-operated rats. Theseresults indicated that retinal IRβ has greater levels of basalphosphorylation than muscle in vehicle treated rats.

The results of experiments quantifying this Western blot analysis areshown in FIG. 2B, presented as a ratio of phosphotyrosine (PY) levels toinsulin receptor beta-subunit (IRβ) amounts (PY/IRβ). In theseexperiments, Western blots interrogated for muscle PY were stripped,reprobed for total IRβ, quantified and the data expressed in terms ofPY/IRβ ratios. The zero time point was set to a ratio of 1. The resultsin FIG. 2B show a nearly 30-fold increase in phosphotyrosine content inmuscle IRβ 15 minutes post-insulin injection, which increase peaked at30 minutes and declined by 60 minutes post-injection. There was asignificant increase in HO phosphorylation with insulin at all pointsexamined. FIG. 2C shows the result obtained in parallel experimentsperformed on retinal tissue. Unlike the results shown in FIG. 2B,tyrosine phosphorylation did not significantly increase in retinaltissue until 30 minutes post injection, was maximal after 45 min andremained elevated 3 4 fold above vehicle-injected controls for 60minutes. IRβ phosphorylation did not change in vehicle injected animals.The discrepancy in the fold increase in retinal tissue may be due to therelatively higher basal IRβ phosphorylation in the retinas of vehicletreated rats, or to the blood-retinal barrier limiting diffusion ofinsulin into the retina.

In addition, 45 min after insulin administration retinal lysates wereanalyzed for Akt activation by Western blotting specifically probed forphosphorylation of Akt (serine 473). These Western blot results, shownin FIG. 2D demonstrated that insulin induced a 48% increase inphosphoserine 473 content of Akt over vehicle injected controls when IRβphosphorylation is maximal (45 minutes post-injection). Quantificationof Western blots, also shown in FIG. 2D indicated there was nostatistical difference between sham and vehicle injected controls. Theseresults indicated that systemic insulin injection can effect changes inIRβ phosphorylation in retina, thereby demonstrating that insulincrosses the blood-retinal barrier. Insulin receptor phosphorylation andactivation occurs in a different temporal manner than muscle, however,and insulin increases Akt phosphorylation in retina.

Thus, even though insulin penetrates the blood-retinal barrier, thedelayed kinetics and lower phosphorylation differential betweenactivated and inactive insulin receptor illustrates the limitations insystemic delivery for affecting retinal diseases related to or dependenton insulin levels, such as diabetic retinopathy.

Example 2

Tyrosine phosphorylation in retinal IRβ and changes therein was comparedwith other insulin responsive tissues under freely fed and moderatelyfasted conditions as follows.

-   -   Six male Sprague-Dawley rats (Charles River) were fasted (18        hours) and were compared to 6 rats that were freely fed        overnight. All rats weighed 200 350 g and were anesthetized with        a 10:1 ketamine:xylazine cocktail as described in Example 1.        Upon loss of motor reflex in the rats' retina, tissue samples        from liver and hindquarter skeletal muscle were obtained,        homogenized and immunoprecipitated as described in Example 1 and        then subjected to Western blot analysis, also as described in        Example 1. The blot in FIG. 3A shows that IRβ phosphorylation        was unaltered in retina, but increased in muscle and liver        compared to fasted rats. Phosphotyrosine-probed immunoblots were        then reprobed for total IRβ content with the immunoprecipitating        antibody as described in Example 1 to normalize the Western blot        data.

The results of these experiments are shown in FIG. 3B as the ratio of PYto total IRβ. Plasma insulin levels were 1.95±0.36 ng/mL in fed ratsversus 0.32±0.049 ng/mL in fasted rats (mean.+−.SEM, p=0.011).Similarly, blood glucose levels were 95.5±2.6 mg/dL in fed rats versus68.83±2.4 mg/dL in fasted rats (mean±SEM, p=0.000008). Image analysis(software from Molecular Dynamics, Sunnyvale, Calif.) of the Westernblots from retinal tissue revealed no differences in IRβ phosphorylationbetween freely fed and fasted rats. In contrast, IRβ phosphorylation inliver and hindquarter skeletal muscle was diminished 23% and 38%,respectively in the fasted rats. On the other hand, IRβ phosphorylationin liver and muscle was significantly greater than in retinal tissue forfreely fed rats.

In summary, retinal IR remained relatively constant in response to thephysiological levels of insulin in circulation. Phosphotyrosine contentof the IRβ in retina did not change, unlike in liver and muscle, despitechanges in nutritional status and physiological increases in circulatinginsulin and blood glucose. IRβ from muscle and liver displayed increasedtyrosine phosphorylation as expected in the freely fed state compared tofasted rats. These results were likely due to the fluctuations ofcirculating insulin in the freely fed condition.

Example 3

Because tyrosine phosphorylation of the IR does not directly measureenzymatic activity, an IR kinase assay was developed.

The IRβ from fasted and fed rats was immunoprecipitated from retina andliver and analyzed by PY immunoblotting for autophosphorylation with (+)and without (−) the addition of ATP to the kinase reaction. IRβimmunoprecipitates obtained as described in Example 2 were subjected toIR kinase assays as follows. The Sepharose bead complex, obtained asdescribed in Example 1, was washed three times with 200 μL of kinasebuffer (50 mM HEPES, pH 7.3, 150 mM NaCl, 20 mM MgCl₂, 2 mM MnCl₂, 0.05%bovine serum albumin, and 0.1% Triton X-100). Western blotting wasperformed on these immunoprecipitates as described in Example 1; theresults of these Western blot experiments (shown below) showed thatwashing the Sepharose bead/immune complex in kinase buffer did notdiminish total bound IRβ. After the last (third) aspiration of kinasebuffer, 500 μL of kinase buffer was added with or without 100 μM ATP(Sigma) to each aliquot of the washed Sepharose bead complex. Theseimmune complexes were then rocked at ambient temperature for 1 minute,and the Sepharose beads then collected by brief centrifugation(approximately 3 seconds). The kinase buffer was aspirated, an equalvolume of 2× Laemmli sample buffer was added and the samples were boiledfor 3 min. SDS-PAGE and phosphotyrosine immunoblotting analysis wasperformed as described in Example 1.

Results of these assays are shown in FIG. 4. The PY immunoblot showedthat retinal IRβ displays tonic tyrosine phosphorylation (− lanes), andthe rate of phosphate incorporated into the IRO is not different betweenfasted and fed rats (+ lanes). Liver IRβ, however, displays less basaltyrosine phosphorylation, which increases in the fed state (− lanes). Asexpected, the rate of IR autophosphorylation is significantly greater inthe freely fed rat liver (+ lanes, p<0.05).

The results of these Western blot experiments shows that the kinasereaction proceeded linearly though 5 minutes of incubation (R²>0.9) andat a non-limiting dose of 100 μM ATP. The time course of IRautophosphorylation continued to proceed in a linear fashion over thecourse of these experiments, and the concentration of ATP used did notlimit the rate of autophosphorylation.

These results further indicated that there was no change in retinal IRβautophosphorylation rate between freely fed and fasted rats. Theseresults also suggested a tonic level of tyrosine phosphorylation andkinase activity despite changes in circulating insulin and glucoselevels. In contrast, in liver and skeletal muscle IRβ phosphorylationwas increased in the freely fed state, leading to a significant increasein autophosphorylation activity. These results thus demonstrated thedifference in insulin signaling physiology within the whole animal.Moreover, these results also indicated that the blood-retinal barriermay discretely regulate insulin flux, unlike in tissues that undergorapid metabolic changes, the results having consequences for insulinadministration in diabetic retinopathy.

Example 4

A tissue explant culture system was used to characterizeinsulin-signaling transduction in retina without the hindrance of theblood retinal barrier present in whole animal models. Such a tissueexplant culture system was developed as follows. Rats as described inExamples 1 and 2 were anesthetized using sodium pentobarbital (7.5mg/kg), and then decapitated upon loss of motor reflexes. Rat retinaswere removed by cutting across the cornea, removing the lens, andsqueezing the eyeball to rapidly extract the retina. Retinas werepre-incubated in MEM (Sigma) supplemented with 5 mM pyruvate and 10 mMHEPES for 15 minutes at 37° C., 5% CO₂, and with gentle shaking. Ten nMinsulin or vehicle was added following pre-incubation. At 2, 5, 15, and30 minutes after insulin addition, retinas were snap-frozen in liquidnitrogen for future use.

To examine the time course of insulin signaling events in the tissue,insulin-treated retinas were compared to retinas that received vehicle(0.9% saline) and to untreated retinas. Tissues were analyzed byimmunoprecipitation and Western blotting as described in Example 1.Western blot data were quantified, as shown in FIG. 5, and revealed asignificant increase in phosphorylation at all time points examined whennormalized to total IRβ. Within 2 minutes of insulin stimulation,retinal IRβ exhibited a nearly 4-fold greater phosphotyrosineimmunoreactivity, which remained elevated at levels 3-fold greater thanbaseline at the 30 minute time point. This analysis demonstrated thatretinal IRβ undergoes increased tyrosine phosphorylation in response toa physiological dose of 10 nM insulin. These results from the explantretina system were similar to the results obtained using a cell culturemodel.

Insulin (10 nM) does not activate the IGF-IRβ in retinal explants.Retinas were treated with either 1.3 nM IGF-1 or 0, 1, 10, 100, and 1000nM insulin, and then retinal lysates were immunoprecipitated with aninsulin-like growth factor I receptor (IGF-IRβ)-specific antibody,followed by PY Western blotting (shown in FIG. 6). The IGF1-R wasimmunoprecipitated and PY Western blotting performed as described above.Insulin at 100 nM produced similar levels of IGF-IRβ phosphorylation aswere found when retinas were contacted with 1.3 nM IGF-1. These resultsdemonstrated that IGF-IRβ phosphorylation remained unchanged in thepresence of 10 nM insulin, but increased with 100 nM insulin. These datafurther indicated that 10 nM insulin does not activate IGF-1Rβ inretinal explants, but has specific effects via the IR. Moreover, ATPlevels over the time course experiments remained constant, indicatingthat the energy status of the tissue was not hindering the insulinsignaling response.

These results suggested that isolated retinal tissue can respondrobustly to physiological insulin concentrations, this responseincluding IRβ tyrosine phosphorylation. The results also suggested thatthe blood-retinal barrier plays a significant role in regulatingcirculating insulin transport since the time course of the response ismuch faster than observed in vivo. Moreover, intraportal insulininjection experiments (Example 1) showed that insulin's access to theretina is limited by the retinal vasculature, the blood-retinal-barrier(BRB). In explanted retina tissue, in contrast, the BRB is essentiallybypassed, and insulin signaling characteristics can be analyzed in ashorter time frame since insulin has direct access to the retina. Aschematic comparison of intraportal insulin injection and explantedretina tissue is shown in FIG. 7.

Example 5

In clinical diabetes, hypoglycemia is the major factor that limits theability of patients to achieve the degree of intensive control ofglycemia needed to prevent or reduce retinopathy. Retinal cells die byapoptosis in diabetes, and insulin is a survival factor for retinalneurons acting via the PI3-kinase/Akt pathway, which is inhibited byhyperglycemia. Systemically administered insulin activates the retinalinsulin receptor, and the activities of the retinal insulin receptor,PI3-kinase and Akt are reduced after 4 weeks of streptozotocin-induceddiabetes. Thus intensive insulin therapy has direct effects on retinalcell survival and function. Since intensive insulin therapy cannot beachieved by most patients due to the hypoglycemic effects of systemicinsulin administration, direct application of insulin to the eye canprovide the survival effects needed to maintain retinal cell health inthe presence of imperfectly controlled systemic diabetes.

To demonstrate the efficacy of the methods of this invention, insulinwas administered directly to the eye via the subconjunctival space ofnormal Sprague-Dawley rats. This route of administration bypasses theBRB for a more direct route of insulin action on retina. By injectingserial dilutions of insulin in this manner, it was discovered that adose of 0.0325 U/100 g insulin activated the IR and Akt kinase pathwaywithout lowering blood glucose values. As shown in FIG. 8, the isphosphorylated in the eye receiving insulin (right column), compared tothe contralateral eye that received vehicle (left column). The retinawas processed for IRβ PY content as described in Example 1. The resultssuggest it is feasible to administer insulin directly to the retina indoses that will not lead to potentially harmful hypoglycemic reactionsand potently activate the insulin receptor.

These results demonstrated the in vivo efficacy of periocular insulinadministration as a treatment for diabetic retinopathy.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that alternatives equivalentthereto are within the spirit and scope of the invention as set forth inthe appended claims.

The invention and the method of making it are now described in suchfull, clear, concise and exact terms as to enable any person skilled inthe art to which it pertains, to make the same. It is to be understoodthat the foregoing describes preferred embodiments of the presentinvention and that modifications may be made therein without departingfrom the spirit or scope of the invention as set forth in the claims.

1. A method of treating a nerve-related vision disorder in a subject inneed thereof comprising the step of administering a therapeuticallyeffective amount of a formulation of an insulinomimetic agent to an eyeaffected by the nerve-related vision disorder via a periocular route,wherein the therapeutically effective amount is effective to achieve alocal therapeutic effect without a substantial systemic effect.
 2. Themethod of claim 1 wherein the route is subconjunctival, sub-Tenon's orintravitreal.
 3. The method of claim 1, wherein the formulation of aninsulinomimetic agent further comprises an augmenting drug.
 4. Themethod of claim 3, wherein the augmenting drug is a thiazolidinedione.5. The method of claim 1, wherein the formulation of the insulinomimeticagent is administered via a solution, a polymeric base, or a pump. 6.The method of claim 1, wherein the formulation of the insulinomimeticagent is administered to the eye by a device implanted therein, whereinthe device releases the insulinomimetic agent at a prescribed rate. 7.The method of claim 1, wherein the insulinomimetic agent is administeredunder the eye's surface membrane.
 8. The method of claim 1, wherein theformulation of the insulinomimetic agent is administered to both eyes ofan individual.
 9. The method of claim 1, wherein the formulation of theinsulinomimetic agent is administered to one or both eyes of an animalusing one or more implanted devices.
 10. The method of claim 1, whereinthe nerve-related vision disorder is diabetic retinopathy.
 11. Themethod of claim 1 wherein the nerve-related vision disorder is a retinaldisorder.
 12. The method of claim 11 wherein the retinal disorder isretinal detachment, retinitis pigmentosa, central retinal arteryocclusion, central retinal vein occlusion, ischemic optic neuropathy,high tension glaucoma, low tension glaucoma, or cataract.
 13. The methodof claim 1 where the periocular route is a non-topical periocular route.14. The method of claim 1 wherein the insulinomimetic agent is aPPAR-gamma activator.